Manipulation of beads in droplets and methods for manipulating droplets

ABSTRACT

Provided herein are methods of splitting droplets containing magnetically responsive beads in a droplet actuator. A droplet actuator having a plurality of droplet operations electrodes configured to transport the droplet, and a magnetic field present at the droplet operations electrodes, is provided. The magnetically responsive beads in the droplet are immobilized using the magnetic field and the plurality of droplet operations electrodes are used to split the droplet into first and second droplets while the magnetically responsive beads remain substantially immobilized.

RELATED APPLICATIONS

This application is a divisional of and claims priority to U.S. patentapplication Ser. No. 15/266,693, entitled “Manipulation of Beads inDroplets and Methods for Manipulating Droplets,” filed Sep. 15, 2016,which is a continuation of and claims priority to U.S. patentapplication Ser. No. 14/978,935, entitled “Manipulation of Beads inDroplets and Methods for Manipulating Droplets,” filed Dec. 22, 2015,now U.S. Pat. No. 9,494,498, issued Nov. 15, 2016, the application ofwhich is a continuation of and claims priority to U.S. patentapplication Ser. No. 14/746,276, entitled “Manipulation of Beads inDroplets and Methods for Manipulating Droplets,” filed Jun. 22, 2015,now U.S. Pat. No. 9,377,455, issued Jun. 28, 2016, the application ofwhich is a continuation of and claims priority to U.S. patentapplication Ser. No. 14/308,110, entitled “Bead Incubation and Washingon a Droplet Actuator” filed Jun. 18, 2014, now U.S. Pat. No. 9,086,345,issued Jul. 21, 2015, the application of which is a divisional of andclaims priority to U.S. patent application Ser. No. 12/761,066, entitled“Manipulation of Beads in Droplets and Methods for ManipulatingDroplets,” filed Apr. 15, 2010, now U.S. Pat. No. 8,809,068, issued Aug.19, 2014, the application of which is A) a continuation of and claimspriority to International Patent Application No. PCT/US2008/080264,entitled “Manipulation of Beads in Droplets,” filed Oct. 17, 2008, whichclaims priority to provisional U.S. patent application Ser. No.60/980,782, entitled “Manipulation of Beads in Droplets,” filed on Oct.17, 2007; and B) a continuation-in-part of and claims priority to U.S.patent application Ser. No. 11/639,531, entitled “Droplet-BasedWashing,” filed Dec. 15, 2006, now U.S. Pat. No. 8,613,889, issued Dec.24, 2013, which claims priority to provisional U.S. Patent ApplicationNos. 60/745,058, entitled “Filler Fluids for Droplet-BasedMicrofluidics” filed on Apr. 18, 2006; 60/745,039, entitled “Apparatusand Methods for Droplet-Based Blood Chemistry,” filed on Apr. 18, 2006;60/745,043, entitled “Apparatus and Methods for Droplet-Based PCR,”filed on Apr. 18, 2006; 60/745,059, entitled “Apparatus and Methods forDroplet-Based Immunoassay,” filed on Apr. 18, 2006; 60/745,914, entitled“Apparatus and Method for Manipulating Droplets with a PredeterminedNumber of Cells” filed on Apr. 28, 2006; 60/745,950, entitled “Apparatusand Methods of Sample Preparation for a Droplet Microactuator,” filed onApr. 28, 2006; 60/746,797 entitled “Portable Analyzer UsingDroplet-Based Microfluidics,” filed on May 9, 2006; 60/746,801, entitled“Apparatus and Methods for Droplet-Based Immuno-PCR,” filed on May 9,2006; 60/806,412, entitled “Systems and Methods for DropletMicroactuator Operations,” filed on Jun. 30, 2006; and 60/807,104,entitled “Method and Apparatus for Droplet-Based Nucleic AcidAmplification,” filed on Jul. 12, 2006; the disclosure of each of theaforementioned patents and patent applications are incorporated hereinby reference in their entirety.

GOVERNMENT INTEREST

This invention was made with government support under DK066956-02 andCA114993-01A2 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND

Droplet actuators are used to conduct a wide variety of dropletoperations. A droplet actuator typically includes two substratesseparated by a gap. The substrates include electrodes for conductingdroplet operations. The space is typically filled with a filler fluidthat is immiscible with the fluid that is to be manipulated on thedroplet actuator. The formation and movement of droplets is controlledby electrodes for conducting a variety of droplet operations, such asdroplet transport and droplet dispensing. There is a need forimprovements to droplet actuators that facilitate handling of dropletswith beads.

SUMMARY OF THE INVENTION

The invention provides a method of dispersing or circulatingmagnetically responsive beads within a droplet in a droplet actuator.The invention, in one embodiment, makes use of a droplet actuator with aplurality of droplet operations electrodes configured to transport thedroplet, and a magnet field present at a portion of the plurality ofdroplet operations electrodes. A bead bead-containing droplet isprovided on the droplet actuator in the presence of the uniform magneticfield. Beads are circulated in the droplet during incubation byconducting droplet operations on the droplet within a uniform region ofthe magnetic field. Other aspects of the invention will be apparent fromthe ensuing description of the invention.

Definitions

As used herein, the following terms have the meanings indicated.

“Activate” with reference to one or more electrodes means effecting achange in the electrical state of the one or more electrodes whichresults in a droplet operation.

“Bead,” with respect to beads on a droplet actuator, means any bead orparticle that is capable of interacting with a droplet on or inproximity with a droplet actuator. Beads may be any of a wide variety ofshapes, such as spherical, generally spherical, egg shaped, disc shaped,cubical and other three dimensional shapes. The bead may, for example,be capable of being transported in a droplet on a droplet actuator orotherwise configured with respect to a droplet actuator in a mannerwhich permits a droplet on the droplet actuator to be brought intocontact with the bead, on the droplet actuator and/or off the dropletactuator. Beads may be manufactured using a wide variety of materials,including for example, resins, and polymers. The beads may be anysuitable size, including for example, microbeads, microparticles,nanobeads and nanoparticles. In some cases, beads are magneticallyresponsive; in other cases beads are not significantly magneticallyresponsive. For magnetically responsive beads, the magneticallyresponsive material may constitute substantially all of a bead or onecomponent only of a bead. The remainder of the bead may include, amongother things, polymeric material, coatings, and moieties which permitattachment of an assay reagent. Examples of suitable magneticallyresponsive beads are described in U.S. Patent Publication No.2005-0260686, entitled, “Multiplex flow assays preferably with magneticparticles as solid phase,” published on Nov. 24, 2005, the entiredisclosure of which is incorporated herein by reference for its teachingconcerning magnetically responsive materials and beads. The fluids mayinclude one or more magnetically responsive and/or non-magneticallyresponsive beads. Examples of droplet actuator techniques forimmobilizing magnetically responsive beads and/or non-magneticallyresponsive beads and/or conducting droplet operations protocols usingbeads are described in U.S. patent application Ser. No. 11/639,566,entitled “Droplet-Based Particle Sorting,” filed on Dec. 15, 2006; U.S.Patent Application No. 61/039,183, entitled “Multiplexing Bead Detectionin a Single Droplet,” filed on Mar. 25, 2008; U.S. patent applicationSer. No. 61/047,789, entitled “Droplet Actuator Devices and DropletOperations Using Beads,” filed on Apr. 25, 2008; U.S. patent applicationSer. No. 61/086,183, entitled “Droplet Actuator Devices and Methods forManipulating Beads,” filed on Aug. 5, 2008; International PatentApplication No. PCT/US2008/053545, entitled “Droplet Actuator Devicesand Methods Employing Magnetically responsive beads,” filed on Feb. 11,2008; International Patent Application No. PCT/US2008/058018, entitled“Bead-based Multiplexed Analytical Methods and Instrumentation,” filedon Mar. 24, 2008; International Patent Application No.PCT/US2008/058047, “Bead Sorting on a Droplet Actuator,” filed on Mar.23, 2008; and International Patent Application No. PCT/US2006/047486,entitled “Droplet-based Biochemistry,” filed on Dec. 11, 2006; theentire disclosures of which are incorporated herein by reference.

“Droplet” means a volume of liquid on a droplet actuator that is atleast partially bounded by filler fluid. For example, a droplet may becompletely surrounded by filler fluid or may be bounded by filler fluidand one or more surfaces of the droplet actuator. Droplets may, forexample, be aqueous or non-aqueous or may be mixtures or emulsionsincluding aqueous and non-aqueous components. Droplets may take a widevariety of shapes; nonlimiting examples include generally disc shaped,slug shaped, truncated sphere, ellipsoid, spherical, partiallycompressed sphere, hemispherical, ovoid, cylindrical, and various shapesformed during droplet operations, such as merging or splitting or formedas a result of contact of such shapes with one or more surfaces of adroplet actuator.

“Droplet Actuator” means a device for manipulating droplets. Forexamples of droplets, see U.S. Pat. No. 6,911,132, entitled “Apparatusfor Manipulating Droplets by Electrowetting-Based Techniques,” issued onJun. 28, 2005 to Pamula et al.; U.S. patent application Ser. No.11/343,284, entitled “Apparatuses and Methods for Manipulating Dropletson a Printed Circuit Board,” filed on filed on Jan. 30, 2006; U.S. Pat.No. 6,773,566, entitled “Electrostatic Actuators for Microfluidics andMethods for Using Same,” issued on Aug. 10, 2004 and U.S. Pat. No.6,565,727, entitled “Actuators for Microfluidics Without Moving Parts,”issued on Jan. 24, 2000, both to Shenderov et al.; Pollack et al.,International Patent Application No. PCT/US2006/047486, entitled“Droplet-Based Biochemistry,” filed on Dec. 11, 2006, the disclosures ofwhich are incorporated herein by reference. Methods of the invention maybe executed using droplet actuator systems, e.g., as described inInternational Patent Application No. PCT/US2007/009379, entitled“Droplet manipulation systems,” filed on May 9, 2007. In variousembodiments, the manipulation of droplets by a droplet actuator may beelectrode mediated, e.g., electrowetting mediated or dielectrophoresismediated.

“Droplet operation” means any manipulation of a droplet on a dropletactuator. A droplet operation may, for example, include: loading adroplet into the droplet actuator; dispensing one or more droplets froma source droplet; splitting, separating or dividing a droplet into twoor more droplets; transporting a droplet from one location to another inany direction; merging or combining two or more droplets into a singledroplet; diluting a droplet; mixing a droplet; agitating a droplet;deforming a droplet; retaining a droplet in position; incubating adroplet; heating a droplet; vaporizing a droplet; condensing a dropletfrom a vapor; cooling a droplet; disposing of a droplet; transporting adroplet out of a droplet actuator; other droplet operations describedherein; and/or any combination of the foregoing. The terms “merge,”“merging,” “combine,” “combining” and the like are used to describe thecreation of one droplet from two or more droplets. It should beunderstood that when such a term is used in reference to two or moredroplets, any combination of droplet operations sufficient to result inthe combination of the two or more droplets into one droplet may beused. For example, “merging droplet A with droplet B,” can be achievedby transporting droplet A into contact with a stationary droplet B,transporting droplet B into contact with a stationary droplet A, ortransporting droplets A and B into contact with each other. The terms“splitting,” “separating” and “dividing” are not intended to imply anyparticular outcome with respect to size of the resulting droplets (i.e.,the size of the resulting droplets can be the same or different) ornumber of resulting droplets (the number of resulting droplets may be 2,3, 4, 5 or more). The term “mixing” refers to droplet operations whichresult in more homogenous distribution of one or more components withina droplet. Examples of “loading” droplet operations includemicrodialysis loading, pressure assisted loading, robotic loading,passive loading, and pipette loading. In various embodiments, thedroplet operations may be electrode mediated, e.g., electrowettingmediated or dielectrophoresis mediated.

“Filler fluid” means a fluid associated with a droplet operationssubstrate of a droplet actuator, which fluid is sufficiently immisciblewith a droplet phase to render the droplet phase subject toelectrode-mediated droplet operations. The filler fluid may, forexample, be a low-viscosity oil, such as silicone oil. Other examples offiller fluids are provided in International Patent Application No.PCT/US2006/047486, entitled, “Droplet-Based Biochemistry,” filed on Dec.11, 2006; and in International Patent Application No. PCT/US2008/072604,entitled “Use of additives for enhancing droplet actuation,” filed onAug. 8, 2008.

“Immobilize” with respect to magnetically responsive beads, means thatthe beads are substantially restrained in position in a droplet or infiller fluid on a droplet actuator. For example, in one embodiment,substantially immobilized beads are sufficiently restrained in positionto permit execution of a splitting operation on a droplet, yielding onedroplet with substantially all of the beads and one dropletsubstantially lacking in the beads.

“Magnetically responsive” means responsive to a magnetic field.“Magnetically responsive beads” include or are composed of magneticallyresponsive materials. Examples of magnetically responsive materialsinclude paramagnetic materials, ferromagnetic materials, ferrimagneticmaterials, and metamagnetic materials. Examples of suitable paramagneticmaterials include iron, nickel, and cobalt, as well as metal oxides,such as Fe.sub.3O.sub.4, BaFe.sub.12O.sub.19, CoO, NiO, Mn.sub.2O.sub.3,Cr.sub.2O.sub.3, and CoMnP.

“Washing” with respect to washing a magnetically responsive bead meansreducing the amount and/or concentration of one or more substances incontact with the magnetically responsive bead or exposed to themagnetically responsive bead from a droplet in contact with themagnetically responsive bead. The reduction in the amount and/orconcentration of the substance may be partial, substantially complete,or even complete. The substance may be any of a wide variety ofsubstances; examples include target substances for further analysis, andunwanted substances, such as components of a sample, contaminants,and/or excess reagent. In some embodiments, a washing operation beginswith a starting droplet in contact with a magnetically responsive bead,where the droplet includes an initial amount and initial concentrationof a substance. The washing operation may proceed using a variety ofdroplet operations. The washing operation may yield a droplet includingthe magnetically responsive bead, where the droplet has a total amountand/or concentration of the substance which is less than the initialamount and/or concentration of the substance. Other embodiments aredescribed elsewhere herein, and still others will be immediatelyapparent in view of the present disclosure.

The terms “top” and “bottom” are used throughout the description withreference to the top and bottom substrates of the droplet actuator forconvenience only, since the droplet actuator is functional regardless ofits position in space.

When a liquid in any form (e.g., a droplet or a continuous body, whethermoving or stationary) is described as being “on”, “at”, or “over” anelectrode, array, matrix or surface, such liquid could be either indirect contact with the electrode/array/matrix/surface, or could be incontact with one or more layers or films that are interposed between theliquid and the electrode/array/matrix/surface.

When a droplet is described as being “on” or “loaded on” a dropletactuator, it should be understood that the droplet is arranged on thedroplet actuator in a manner which facilitates using the dropletactuator to conduct one or more droplet operations on the droplet, thedroplet is arranged on the droplet actuator in a manner whichfacilitates sensing of a property of or a signal from the droplet,and/or the droplet has been subjected to a droplet operation on thedroplet actuator.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a top view of a portion of a droplet actuator usefulfor incubating a droplet including magnetically responsive beads;

FIG. 2 illustrates a top view of a portion of a droplet actuator usefulfor incubation of antibodies, wherein a sample and magneticallyresponsive beads are provided within the magnet field of a magnet;

FIG. 3 illustrates a top view of a portion of a droplet actuator usefulfor incubation of magnetically responsive beads within a droplet,wherein a sample and magnetically responsive beads are subjected todroplet operations within the magnet field of a magnet;

FIG. 4 illustrates a method of shielding the effect of multiple magnetsin a droplet actuator by using a magnetic shielding material;

FIG. 5 illustrates a magnet array for performing multiple immunoassays;

FIG. 6 illustrates simulation results that show the surface field of 2columns of the magnet array of FIG. 5;

FIG. 7 illustrates a top view of a portion of a droplet actuator usefulfor resuspension of beads (e.g., magnetically-responsive beads) within areservoir configured with multiple electrodes;

FIG. 8 illustrates a shows a top view of a portion of a droplet actuatoruseful for resuspending beads (e.g., magnetically-responsive beads)within a reservoir by pushing out a finger of liquid and then mergingback;

FIG. 9 illustrates a top view of a portion of the droplet actuator ofFIG. 8 including a reservoir in which beads are resuspended by applyinghigh frequency voltage to the reservoir electrode;

FIG. 10 illustrates a side view of a droplet actuator that includes atop substrate and bottom substrate separated by a gap;

FIG. 11 illustrates a side view of another embodiment of a dropletactuator including a top substrate and bottom substrate separated by agap;

FIG. 12 illustrates a side view of yet another embodiment of a dropletactuator that includes a top substrate and bottom substrate separated bya gap;

FIG. 13 illustrates a top view of a portion of a droplet actuator usefulfor a process of asymmetrically splitting a droplet;

FIG. 14 illustrates a top view of a portion of a droplet actuator usefulfor a process employing a hydrophilic patch in a droplet splittingoperation;

FIG. 15 illustrates a top view of a portion of a droplet actuator usefulfor a process of using a magnetic strip that is integrated into thegasket material at the point of bead immobilization;

FIG. 16 illustrates a side view of a droplet actuator that includes atop substrate and bottom substrate that are separated by a gap usefulfor facilitating consistent droplet splitting by use of a physicalbarrier in the droplet actuator;

FIG. 17 illustrates a side view of the portion of droplet actuator inFIG. 16 useful for facilitating consistent droplet splitting by use of amagnetic physical barrier in the droplet actuator;

FIG. 18 illustrates embodiments of electrode configuration for improveddroplet splitting; and

FIG. 19 illustrates detection strategies for quantifying an analyte.

DESCRIPTION

The invention provides droplet actuators having specializedconfigurations for manipulation of droplets including beads and/or formanipulation of beads in droplets. In certain embodiments, the dropletactuators of the invention include magnets and/or physical barriersmanipulation of droplets including beads and/or for manipulation ofbeads in droplets. The invention also includes methods of manipulatingof droplets including beads and/or for manipulation of beads indroplets, as well as methods of making and using the droplet actuatorsof the invention. The droplet actuators of the invention are useful for,among other things, conducting assays for qualitatively and/orquantitatively analyzing one or more components of a droplet. Examplesof such assays include affinity based assays, such as immunoassays;enzymatic assays; and nucleic acid assays. Other aspects of theinvention will be apparent from the ensuing discussion.

7.1 Incubation of Beads

In certain embodiments, the invention provides droplet actuators andmethods for incubating beads. For example, a sample includingbead-containing antibodies may be incubated on the droplet actuator inorder to permit one or more target components to bind to the antibodies.Examples of target components include analytes; contaminants; cells,such as bacteria and protozoa; tissues; and organisms, such asmulticellular parasites. In the presence of a magnet, magnetic beads inthe droplet may be substantially immobilized and may fail to circulatethroughout the droplet. The invention provides various dropletmanipulations during incubation of droplets on a droplet actuator inorder to increase circulation of beads within the droplet and/orcirculation of droplet contents surrounding beads. It will beappreciated that in the various embodiments described below employingmagnetically responsive beads, beads that are not substantiallymagnetically responsive may also be included in the droplets.

FIG. 1 illustrates techniques that are useful process of incubating adroplet including magnetically responsive beads. Among other things, thetechniques are useful for enhancing circulation of fluids and beadswithin the droplet during an incubation step.

In FIG. 1, each step is illustrated on a path of electrodes 110. Amagnet 112 is associated with a subset of electrodes 110. Magnet 112 isarranged relative to the electrodes 110 such that a subset of electrodes110 are within a uniform region of the magnetic field produced by magnet112. Bead clumping is reduced when the droplet is present in thisuniform region.

In Step 1, droplet 116 is located atop magnet 112. Beads 116 aresubstantially immobilized in a distributed fashion adjacent to thedroplet operations surface. The beads are generally less clumped thanthey would be in the presence of a non-uniform region of the magneticfield. In Step 2 droplet 114 is split using droplet operations into twosub-droplets 114A, 114B. During the splitting operation beads and liquidare circulated within the droplets 114, 114A and 114B. In Step 3Droplets 114A and 114B are merged using droplet operations into a singledroplet 114. This merging operation is accomplished within the uniformregion of the magnetic field. During the merging operation beads andliquid are further circulated within the droplets 114, 114A and 114B.

In Step 4, droplet 114 is transported using droplet operations alongelectrodes 110 away from the magnet 112. As droplet 116 moves away frommagnet 110, beads 116 are pulled to the edge of droplet 114 that nearestthe magnet 112. Movement of beads 116 within droplet 114 providesfurther beneficial circulation of beads and liquid within the droplet114. In Step 5, droplet 116 is transported using droplet operations backto the step 1 position. Beads 116 within the droplet 116 are againdispersed in the presence of the uniform magnetic field of magnet 112.This redistribution of beads, as droplet 114 returns to its positionwithin the uniform region of the magnetic field provides furtherbeneficial circulation of beads and liquid within the droplet 114.

These steps may be conducted in any logical order. Each step may beconducted any number of times between the other steps. For example,Steps 1-3 may be repeated multiple times before moving onto Step 4.Similarly, Steps 3-5 may be repeated multiple times before returning toSteps 1-3. Moreover, all steps are not required. For example, in oneembodiment, an incubation step in an assay is accomplished by repeatingSteps 1-3. In another embodiment, an incubation step in an assay isaccomplished by repeating Steps 3-5.

The incubation method of the invention is useful for enhancingcirculation of magnetically responsive beads with the liquid in adroplet while the droplet remains in the presence of a magnetic field.Among other advantages, the approach may reduce bead clumping and permittighter droplet actuator designs making more efficient use of dropletactuator real estate.

In one embodiment, the invention provides a droplet operationsincubation scheme, that does not allow magnetically responsive beads tobe introduced into a region of the magnetic field which is sufficientlynon-uniform to cause bead clumping. In another embodiment, the inventionprovides a merge-and-split incubation scheme, that does not allowmagnetically responsive beads to be introduced into a region of themagnetic field which is sufficiently non-uniform to cause bead clumping.In yet another embodiment, the invention provides a droplet transportincubation scheme, that does not allow magnetically responsive beads tobe introduced into a region of the magnetic field which is sufficientlynon-uniform to cause bead clumping.

Any combination of droplet operations which result in effective mixing(e.g., substantially complete mixing) may be chosen. Mixing is completewhen it is sufficient for conducting the analysis being undertaken. Thedroplet may be oscillated in the presence of the uniform region of themagnetic field by transporting the droplet back and forth within theuniform region. In some cases, electrode sizes used for the oscillationmay be varied to increase circulation within the droplet. In some cases,droplet operations electrodes are used to effect droplet operations totransport a droplet back and forth or in one or more looping patterns.Preferably the oscillation pattern does not allow to be introduced intoa region of the magnetic field which is sufficiently uniform to causebead clumping.

In some cases, droplet operations are performed at an edge of the magnetto more equally redistribute the magnetically responsive beads. In somecases, droplet operations are performed away from the magnet, followedby transporting the droplet.

FIG. 2 illustrates another process of incubation of antibodies, whereina sample and magnetically responsive beads are provided within themagnet field of a magnet, e.g., within a uniform magnetic field regionof a magnet. FIG. 2 shows a top view of a portion of droplet actuator100 that is described in FIG. 1.

In Step 1, beads 116 are substantially immobilized along the surface ofthe droplet operations electrodes 110 due to the magnetic field of themagnet 112. I Step 2, droplet 114 is split using droplet operations intotwo droplets 118, both remaining in the uniform region of the magneticfield. In step 4, the two droplets 118 are transported away from themagnet 112, thereby attracting the beads 116 to the edge of the twodroplets 118 nearest the magnet 112. This operation causes flow reversalwithin the droplets 118, which enhances effective mixing. The twodroplets 118 may alternatively be transported away from the magnet indifferent directions, such as in opposite directions. In Step 4 the twodroplets 118 are merged into one droplet 116. In step 5, the droplet 116is transported back to the step 1 position, causing the beads 116 todisperse within the droplet 116.

FIG. 3 illustrates another process of incubation of magneticallyresponsive beads within a droplet, wherein a sample and magneticallyresponsive beads are subjected to droplet operations within the magnetfield of a magnet. FIG. 3 shows a top view of a portion of a dropletactuator 300 that includes a set of droplet operations electrodes 310(e.g., electrowetting electrodes) that is arranged in sufficientproximity to a magnet, such that a droplet 314 moving along the dropletoperations electrodes 310 is within the magnet field of the magnet,e.g., a region of uniform magnetic field. For example, the set ofdroplet operations electrodes 310 are arranged in a closed loop and inthe presence of two magnets, such as a magnet 312A and magnet 312B, asshown in FIG. 3. In this embodiment, the droplet 314 may include sampleand beads 316, and some or all of the beads may be magneticallyresponsive.

In Step 1, sample with beads 316 in the droplet 314 is provided ondroplet actuator. Beads 316 are substantially immobilized along thesurface of the droplet operations electrodes 310 due to the magneticfield of the first magnet 312A that is located at “lane A” of theelectrode loop. In Step 2, the droplet 314 is split using dropletoperations into two droplets 318, distributing the beads 316 in the twodroplets 318 at “lane A” of the electrode loop. In Step 3, the twodroplets 318 are transported using droplet operations in oppositedirections away from the first magnet 312A at “lane A” and toward thesecond magnet 312B that is located at “lane B” of the electrode loop. InStep 4, in the presence of the second magnet 312B at “lane B,” droplets318 are merged into one droplet 320.

In Steps 5-6, not shown, the process of steps 1-3 may be essentiallyrepeated in reverse. In step 5, droplet 320 may be split into twodroplets 318, distributing the beads 316 in the two droplets 318 at“lane B.” In Step 6, droplets 318 are transported in opposite directionsaway from the second magnet 312B at “lane B” and back to the firstmagnet 312A at “lane A.” In Step 7, in the presence of the first magnet312A at “lane A,” droplets 318 are merged into one droplet 320.

The droplet split and merge operation as described above provideefficient dispersion of beads in the presence of a magnet, therebyimproving the efficiency of the binding of antibodies and the analyte.The various droplet operations may be conducted in primarily orcompletely in uniform regions of the magnetic fields generated bymagnets 312A, 312B. Alternatively, the droplet split and merge operationas described above may be performed away from the magnet and/or near theedge of the magnet.

7.2 Magnet Configurations

FIG. 4 illustrates a method of shielding the effect of multiple magnetsin a droplet actuator 400 by using a magnetic shielding material,preferably one that has high magnetic permeability. One example of suchmaterial is Mu-metal foil. Mu-metal is a nickel-iron alloy (e.g., 75%nickel, 15% iron, plus copper and molybdenum) that has very highmagnetic permeability. FIG. 4 shows a top view of multiple washing lanes410, wherein each washing lane 410 includes a string of dropletoperations electrodes 412 in the presence of a magnet 414. An electrodearray 416 (e.g., an array of electrowetting electrodes) for performingdroplet operations feed the multiple washing lanes 410. Additionally,the droplets 418 that are transported may include magneticallyresponsive beads (not shown). Furthermore, this embodiment provides amagnetic shield 420, provided as a layer that is beneath the electrodearray 416.

Because of the presence of multiple magnets 414, which are used toimmobilize magnetically responsive beads during washing, themagnetically responsive beads in the reservoir tend to becomeaggregated, sometimes irreversibly. When bead-containing droplets aredispensed using droplet operations, bead aggregation may cause thenumber of beads that are present in each dispensed droplet to vary.Variation in bead dispensing may affect the assay result, which is notdesirable. The invention, as shown in FIG. 4, provides magnetic shield420 in the area under the electrode array 416 of the droplet actuator400. The magnetic shield 420 may be formed of alloys, such as Mu-metalfoil, which shields the magnetically responsive beads within theelectrode array 416 from stray magnetic fields 422.

FIG. 5 illustrates a magnet array 500 for performing multipleimmunoassays that has reduced, preferably substantially no, interferencedue to adjacent magnets within a droplet actuator (not shown) having asubstrate associated with droplet operations electrodes. The electrodesare arranged for conducting one or more droplet operations on a dropletoperations surface of the substrate. Magnets, such as the magnet array500 shown in FIG. 5, may be arranged with respect to the dropletactuator such that one or more magnets cancels out some portion of amagnetic field of one or more other magnets. In this manner, an area ofthe surface may have some portions that are subject to magnetic fieldsand some portions in which the magnetic fields have been cancelled out.For example, magnets may be arranged to cancel the field in areas of thedroplet actuator that includes liquid along with magnetically responsivebeads. Specifically reservoirs, incubation regions, detection regionsare preferably in regions in which the magnetic fields have beencancelled out.

In one embodiment, the arrangement involves an array of alternatelyplaced magnets, e.g., as shown in FIG. 5. In general, magnets may belocated in any position which supplies a magnetic field to the vicinityof the droplet operations surface where the magnetic field is desiredand eliminates or weakens the magnetic field in other areas where themagnetic field is not desired. In one embodiment, a first magnetproduces a first magnetic field where it is desirable to immobilizemagnetically responsive beads in a droplet, while a second magnetproduces a second magnetic field which cancels or weakens a portion ofthe first magnetic field. This arrangement produces a device in which aportion of the droplet operations surface that would have otherwise beeninfluenced by the first magnetic field is subjected to a weak or absentfield because the first magnetic field has been cancelled or weakened bythe second magnetic field.

In one embodiment, one or more of the magnets is fixed in relation tothe droplet operations surface, and the invention comprises conductingone or more droplet operations using droplets that contain magneticallyresponsive beads, where the droplets are in proximity to one or moremagnets and are in the presence or absence of a magnetic field.

In another embodiment, the magnetic field exerts sufficient influenceover magnetically responsive beads that the droplets may besubstantially immobile during one droplet operation, such as a splittingoperation, and yet not so stable that the droplets are restrained frombeing transported away from the magnetic field with the magnet. In thisembodiment, the droplet may be surrounded by a filler fluid, and yet thedroplet with the magnetically responsive beads may be transported awayfrom the magnetic with substantially no loss of magnetically responsivebeads to the filler fluid.

FIG. 6 illustrates simulation results 600 that show the surface field of2 columns of magnet array 500 of FIG. 5.

7.3 Resuspension of Beads within a Reservoir

FIG. 7 illustrates a process of resuspension of beads (e.g.,magnetically-responsive beads) within a reservoir configured withmultiple electrodes within the. FIG. 7 shows a top view of a portion ofa droplet actuator 700 that includes a reservoir 710 that is formed ofmultiple electrodes (e.g., electrodes 1 through 9 in a 3×3 array),whereby the reservoir 710 feeds a line of droplet operations electrodes712 (e.g., electrowetting electrodes) to which droplets that containbeads may be dispensed.

Referring to FIG. 7, a process of resuspension of beads within areservoir by having multiple electrodes within the same reservoir mayinclude, but is not limited to, the following steps. In Step 1, beads714 are aggregated within the solution 716 due to the presence ofmultiple magnets (not shown). In Step 2, electrodes within the reservoir710 are used to subject the solution 716 to droplet operations, therebyresuspension of the beads 714. The electrode activation sequence may berandomized to create more chaotic flow fields for more efficientresuspension. The liquid may be split and merged and subjected to otherdroplet operations.

During the above-described process, the electrode activation sequencemay be chosen such that the beads are mixed well by means of dropletoperations. Additionally, when dispensing (e.g., pulling out a finger offluid) a bead droplet from the electrode array of the reservoir, all theelectrodes within the reservoir may be switched ON and OFF at the sametime, depending on the requirement. It should be noted that an almostinfinite variety of electrode shapes is possible. Any shape which iscapable of facilitating a droplet operation will suffice.

The resuspension process may be repeated between every 1, 2, 3, 4, 5 ormore droplet dispensing operations. The resuspend-and-dispense patternmay be adjusted as required based on the specific characteristics ofbead types and droplet compositions. For example, in one embodiment, theprocess of the invention results in dispensing bead-containing dropletswith greater than 95% consistency in bead count. In another embodiment,the process of the invention results in dispensing bead-containingdroplets with greater than 99% consistency in bead count. In anotherembodiment, the process of the invention results in dispensingbead-containing droplets with greater than 99.9% consistency in beadcount. In another embodiment, the process of the invention results indispensing bead-containing droplets with greater than 99.99% consistencyin bead count.

FIG. 8 illustrates a process of resuspending beads (e.g.,magnetically-responsive beads) within a reservoir by pushing out afinger of liquid and then merging back. FIG. 8 shows a top view of aportion of a droplet actuator 800 that includes a reservoir 810 thatfeeds a line of droplet operations electrodes 812 (e.g., electrowettingelectrodes) to which droplets that contain beads may be dispensed.Additionally, the reservoir includes a solution 814 that includes beads816. Referring to FIG. 8, a process of resuspension of beads within areservoir by pushing out a finger of liquid and then merging back mayinclude, but is not limited to, the following steps.

In Step 1, beads 816 are aggregated within the solution 814 due to thepresence of multiple magnets (not shown). In Step 2, a finger ofsolution 814 that includes beads 816 is pulled out of the reservoir 810using droplet operations. In Step 3, a 2X slug 818 is dispensed bysplitting the middle of the finger of solution 814. In Step 4, the 2Xslug 818 is merged back with the solution 814 that includes magneticallyresponsive beads 816 within the reservoir 810.

Steps 2 through 4 may be repeated until the desired degree ofresuspension is achieved, e.g., until substantially completelyresuspended beads are obtained within the bead solution of the reservoir810. When the desired degree of resuspension is achieved,bead-containing droplets may be dispensed, achieving a target percentageof variation in each droplet.

The resuspension process may be repeated, between every 1, 2, 3, 4, 5 ormore droplet dispensing operations. The resuspend-and-dispense patternmay be adjusted as required based on the specific characteristics ofbead types and droplet compositions. For example, in one embodiment, theprocess of the invention results in dispensing bead-containing dropletswith greater than 95% consistency in bead count. In another embodiment,the process of the invention results in dispensing bead-containingdroplets with greater than 99% consistency in bead count. In anotherembodiment, the process of the invention results in dispensingbead-containing droplets with greater than 99.9% consistency in beadcount. In another embodiment, the process of the invention results indispensing bead-containing droplets with greater than 99.99% consistencyin bead count.

FIG. 9 illustrates a reservoir in which beads are resuspended byapplying high frequency voltage to the reservoir electrode. The figureshows a top view of a portion of droplet actuator 800 of FIG. 8.Reservoir 810 includes a droplet 814 that includes magneticallyresponsive beads 816. Beads 816 in a reservoir 810 may tend to becomeaggregated due to, for example, the presence of nearby magnets (notshown). Aggregation may adversely affect bead count in dispensed beads,adversely impacting reliability of assay results for assays conductedusing the dispensed bead-containing droplets. Beads 816 may beresuspended within the magnetically responsive bead solution within thereservoir 810 by applying a high frequency AC voltage to the reservoirelectrode 810, in accordance with the invention. Because of the highfrequency AC voltage, the magnetically responsive beads 816 tend tooscillate because of the wetting and dewetting of the contact line ofthe droplet. This oscillation at the periphery disperses themagnetically responsive beads 816 and resuspends them in thesupernatant. In one example, the high frequency AC voltage may be in therange from about 100 volts to about 300 volts with a frequency fromabout 10 Hz to about 1000 Hz.

The resuspension process may be repeated between every 1, 2, 3, 4, 5 ormore droplet dispensing operations. The resuspend-and-dispense patternmay be adjusted as required based on the specific characteristics ofbead types and droplet compositions. For example, in one embodiment, theprocess of the invention results in dispensing bead-containing dropletswith greater that 95% consistency in bead count. In another embodiment,the process of the invention results in dispensing bead-containingdroplets with greater than 99% consistency in bead count. In anotherembodiment, the process of the invention results in dispensingbead-containing droplets with greater than 90.9% consistency in beadcount. In another embodiment, the process of the invention results indispensing bead-containing droplets with greater than 90.99% consistencyin bead count.

7.4 Improving Dispersion of Magnetically Responsive Beads by MagnetConfigurations

FIG. 10 illustrates a side view of a droplet actuator 1000 that includesa top substrate 1010 and bottom substrate 1012 that are separated by agap. A set of droplet operations electrodes 1014 (e.g., electrowettingelectrodes) is provided on the bottom substrate 1012. Additionally, afirst electromagnet 1016 is arranged near the top substrate 1010 and asecond electromagnet 1018 is arranged near the bottom substrate 1012.The proximity of the electromagnets 1016 and 1018 to the dropletactuator 1000 is sufficiently close that the gap is within the magneticfields thereof. A droplet 1020 that includes magnetically responsivebeads 1022 is in the gap and may be manipulated along the dropletoperations electrodes 1014.

Electromagnets 1016 and 1018 may be used to improve dispersion ofmagnetically responsive beads 1022. Improved dispersion may, forexample, improve binding efficiency of antibodies and analytes to thesurface of the beads. By providing an electromagnet on the top andbottom of the droplet 1020, the magnetically responsive beads 1022 maybe effectively dispersed within the droplet 1020 by switching ON and OFFthe magnetic fields of electromagnets 1016 and 1018. In one example,FIG. 10A shows the electromagnet 1016 turned ON and the electromagnet1018 turned OFF, which causes the beads 1022 to be attracted to theelectromagnet 1016 and are, therefore, pulled to the electromagnet 1016side of the droplet 1020. Subsequently, electromagnet 1018 is turned ONand electromagnet 1016 is turned OFF, which causes the beads 1022 to beattracted to electromagnet 1018 and are, therefore, pulled to theelectromagnet 1018 side of the droplet 1020. Alternating the activationof electromagnets 1016 and 1018 may be repeated until resuspension ofthe beads 1022 is substantially achieved. FIG. 10B shows bothelectromagnets 1016 and 1018 turned ON at the same time, which causes apillar of beads 1022 to form through droplet 1020. Various changes inthe configuration of magnet activation (ON/ON, ON/OFF, OFF/ON, andOFF/OFF) may be used to circulate magnetically responsive beads 1022within droplet 1020. In some embodiments, the pattern of magnetactivation may be randomized. Examples include ON/OFF, OFF/ON, ON/OFF,OFF/ON, ON/OFF, etc.; ON/ON, ON/OFF, OFF/ON, ON/ON, ON/OFF, OFF/ON,ON/ON, ON/OFF, OFF/ON, etc; ON/ON, ON/OFF, OFF/ON, OFF/OFF, ON/ON,ON/OFF, OFF/ON, OFF/OFF, ON/ON, ON/OFF, OFF/ON, OFF/OFF, etc.; ON/OFF,OFF/OFF, OFF/ON, OFF/OFF, ON/OFF, OFF/OFF, OFF/ON, OFF/OFF, ON/OFF,OFF/OFF, OFF/ON, OFF/OFF, etc. Various other magnet activation patternswill be apparent to one of skill in the art in light of the presentspecification.

FIG. 11 illustrates a side view of a droplet actuator 1100 including atop substrate 1110 and bottom substrate 1112 that are separated by agap. A set of droplet operations electrodes 1114 (e.g., electrowettingelectrodes) is provided on the bottom substrate 1112. Additionally,multiple magnets 1116 are arranged near the top substrate 1110 andmultiple magnets 1116 are arranged near the bottom substrate 1112. Inone example, magnets 1116-1, 1116-3, and 1116-5 are arranged near thetop substrate 1110 and magnets 1116-2, 1116-4, and 1116-6 are arrangednear the bottom substrate 1112. The proximity of the magnets 1116 to thedroplet actuator 1100 is sufficiently close that the gap is within themagnetic fields thereof. A slug of liquid 1118 (e.g., antibodies samplemixture) that includes magnetically responsive beads 1120 is in the gapalong the droplet operations electrodes 1114. This aspect of theinvention may improve the binding of analytes or other targetsubstances, such as cells, with antibodies that are present on the beads1120.

Referring to FIG. 11, a process of providing improved dispersion ofmagnetically responsive beads by use of a magnet arrangement, such asshown in FIG. 11, may include, but is not limited to, the followingsteps.

Step 1: Magnet 1116-1=OFF, magnet 1116-2=0N, magnet 1116-3=OFF, magnet1116-4=OFF, magnet 1116-5=OFF, and magnet 1116-6=OFF, which causes themagnetically responsive beads 1120 to be attracted toward magnet 1116-2.

Step 2: Magnet 1116-1=OFF, magnet 1116-2=OFF, magnet 1116-3=0N, magnet1116-4=OFF, magnet 1116-5=OFF, and magnet 1116-6=OFF, which causes themagnetically responsive beads 1120 to be attracted toward magnet 1116-3.

Step 3 (not shown): Magnet 1116-1=OFF, magnet 1116-2=OFF, magnet1116-3=OFF, magnet 1116-4=OFF, magnet 1116-5=OFF, and magnet 1116-6=0N,which causes the magnetically responsive beads 1120 to be attractedtoward magnet 1116-6.

Step 4 (not shown): Magnet 1116-1=OFF, magnet 1116-2=OFF, magnet1116-3=OFF, magnet 1116-4=OFF, magnet 1116-5=0N, and magnet 1116-6=OFF,which causes the magnetically responsive beads 1120 to be attractedtoward magnet 1116-5.

Step 5 (not shown): Magnet 1116-1=OFF, magnet 1116-2=OFF, magnet1116-3=OFF, magnet 1116-4=0N, magnet 1116-5=OFF, and magnet 1116-6=OFF,which causes the magnetically responsive beads 1120 to be attractedtoward magnet 1116-4.

Step 6 (not shown): Magnet 1116-1=0N, magnet 2=OFF, magnet 3=OFF, magnet4=OFF, magnet 5=OFF, and magnet 6=OFF, which causes the magneticallyresponsive beads to be attracted toward magnet 1.

Steps 1 through 6 may be repeated until a desired degree of dispersionor circulation of magnetically responsive beads 1120 and liquid isachieved.

FIG. 12 illustrates a side view of a droplet actuator 1200 that includesa top substrate 1210 and bottom substrate 1212 that are separated by agap. A set of droplet operations electrodes 1214 (e.g., electrowettingelectrodes) is provided on the bottom substrate 1212. A droplet 1216that includes magnetically responsive beads 1218 is provided in the gapand may be manipulated along the droplet operations electrodes 1214.Additionally, a first magnet 1220A is arranged near the top substrate1210 and a second magnet 1220B is arranged near the bottom substrate1212. The proximity of the magnets 1220A and 1220B to the dropletactuator 1200 is sufficiently close that the gap is within the magneticfields thereof. However, the distance of the magnets 1220A and 1220Bfrom the droplet actuator 1200 may be adjusted by, for example, amechanical means, thereby adjusting the influence of the magnetic fieldsupon the magnetically responsive beads 1218.

Mechanical movement of the magnets 1220A and 1220B disperses orotherwise circulates magnetically responsive beads and liquids withinthe droplet. In one example, FIG. 12A shows both magnets 1220A and 1220Bin close proximity to the droplet actuator 1200, which causes a pillarof beads 1218 to form through the droplet 1216. In another example, FIG.12B shows the magnet 1220A only may be moved mechanically by a distance“x” where substantially no magnetic field of magnet 1220A reaches themagnetically responsive beads 1218 and, thus, the beads 1218 areattracted toward the magnet 1220B, thereby dispersing the beads 1218. Inlike manner, the magnet 1220B only may be moved mechanically by adistance “x” where substantially no magnetic field of magnet 1220Breaches the magnetically responsive beads 1218 and, thus, the beads areattracted toward the first magnet 1220A, thereby dispersing the beads1218. By, for example, alternating the mechanical movement of themagnets, effective dispersion of magnetically responsive beads 1218 issubstantially ensured. In some embodiments, both magnets are moved.Magnets may be oscillated to rapidly circulate beads and liquids withinthe droplet.

7.5 Improved Droplet Splitting by Magnet Configurations

FIG. 13 illustrates a process of asymmetrically splitting a droplet.FIG. 13 shows a top view of a portion of a droplet actuator 1300 thatincludes a set of droplet operations electrodes 1310 (e.g.,electrowetting electrodes) that is arranged in sufficient proximity to amagnet 1312, such that a droplet 1314 moving along the dropletoperations electrodes 1310 is within the magnet field of the magnet1312, e.g., a region of uniform magnetic field. In this embodiment, thedroplet 1314 may be may include sample and beads 1316, and some or allof the beads 1316 may be magnetically responsive.

The process may include, without limitation, the following steps. Instep 1, after immobilizing the magnetically responsive beads 1316 to alocalized area in the presence of magnet 1312, droplet operationselectrodes 1310 are activated to extend droplet 1314 into a 4x-slug ofliquid that extends beyond the boundary of magnet 1312. In Step 2,droplet operations electrode 1310 is deactivated, and the next twodroplet operations electrodes 1310 remain on, and a third dropletoperations electrode is activated to provide the asymmetric split. Theprocess may, for example, be employed in a merge-and-split bead washingprotocol.

FIG. 14 illustrates a process employing a hydrophilic patch in a dropletsplitting operation. FIG. 14 shows a top view of a portion of a dropletactuator 1400 that includes a set of droplet operations electrodes 1410(e.g., electrowetting electrodes) arranged in sufficient proximity to amagnet 1412, such that a droplet moving along the droplet operationselectrodes 1410 is within the magnet field of the magnet 1412, e.g., aregion of uniform magnetic field. In this embodiment, the droplet may bemay include sample and beads 1414, and some or all of the beads may bemagnetically responsive.

The process may include, without limitation, the following steps. InStep 1, a small hydrophilic patch 1416, which is patterned on the topsubstrate (not shown) and opposite a certain droplet operationselectrode 1410, immobilizes the aqueous slug 1418, and the magnet 1412immobilizes the magnetically responsive beads 1414. In Step 2, a dropletsplitting operation is executed (e.g., forming droplets 1420 and 1422).The hydrophilic patch 1416 ensures droplet splitting at the same pointin relation to the droplet operations electrode 1410 that is downstreamof the hydrophilic patch 1416. In this example, the magneticallyresponsive beads 1414 remain substantially immobilized in droplet 1422by the magnet 1412 and droplet 1522 is substantially free of beads 1420.The process may, for example, be employed in a merge-and-split beadwashing protocol.

FIG. 15 illustrates a process of using a magnetic strip that isintegrated into the gasket material at the point of bead immobilization.FIG. 15 shows a top view of a portion of a droplet actuator 1500 thatincludes a set of droplet operations electrodes 1510 (e.g.,electrowetting electrodes) that is arranged in sufficient proximity to amagnetic strip 1512 that is integrated into the gasket material 1514 ofthe droplet actuator 1500, such that a droplet moving along the dropletoperations electrodes 1510 is within the magnet field of the magneticstrip 1512, e.g., a region of uniform magnetic field. In thisembodiment, the droplet may be may include sample and beads 1516, andsome or all of the beads may be magnetically responsive.

The process may include, but is not limited to, the following steps. InStep 1, magnetic strip 1512 immobilizes the magnetically responsivebeads 1516 in an aqueous slug 1518. In Step 2, a droplet splittingoperation occurs (e.g., forming droplets 1520 and 1522), whereby themagnetically responsive beads 1516 remain substantially immobilized indroplet 1520 by the magnetic strip 1512 and droplet 1522 issubstantially free of beads 1516. The process may, for example, beemployed in a merge-and-split bead washing protocol.

7.6 Improved Droplet Splitting by Physical Barrier

FIG. 16 illustrates a process of facilitating consistent dropletsplitting by use of a physical barrier in the droplet actuator. FIG. 16shows a side view of a droplet actuator 1600 that includes a topsubstrate 1610 and bottom substrate 1612 that are separated by a gap. Aset of droplet operations electrodes 1614 (e.g., electrowettingelectrodes) is provided on the bottom substrate 1612. Additionally, amagnet 1616 is arranged in sufficient proximity to the dropletoperations electrodes 1614, such that a droplet moving along the dropletoperations electrodes 1610 is within the magnet field of the magnet1616, e.g., a region of uniform magnetic field. In this embodiment, thedroplet may be may include sample and beads 1618, and some or all of thebeads 1618 may be magnetically responsive. Additionally, the dropletactuator 1600 includes a physical barrier 1620 that is arranged as shownin FIG. 16. The physical barrier 1620 is used to reduce the gap at thepoint of splitting, thereby assisting the droplet splitting operation.Additionally, because of the existence of the rigid barrier, consistentsplitting may be obtained substantially at the same point. Further, thephysical barrier 1620 may in some cases substantially nonmagnetic.

The process may include, but is not limited to, the following steps. InStep 1, magnet 1612 immobilizes the magnetically responsive beads 1618in, for example, an aqueous slug 1622. The aqueous slug 1622 isintersected by the physical barrier 1620, which reduces the gap. In Step2, a droplet splitting operation occurs (e.g., forming droplets 1624 and1626), whereby the magnetically responsive beads 1618 remainsubstantially immobilized by the magnet 1616 and the physical barrier1620 is used to reduce the gap at the point of splitting, therebyassisting the droplet splitting operation. In this example, magneticallyresponsive beads 1618 remain substantially immobilized in droplet 1624by the magnet 1612 and droplet 1626 is substantially free of beads 1618.For example, substantially all of the magnetically responsive beads 1618may remain in droplet 1618, while droplet 1610 may be substantially freeof magnetically responsive beads 1618. The process may, for example, beemployed in a merge-and-split bead washing protocol.

FIG. 17 illustrates a process of facilitating consistent dropletsplitting by use of a magnetic physical barrier in the droplet actuator.FIG. 17 shows a side view of the portion of droplet actuator 1600 thatis described in FIG. 16. However, FIG. 17 shows that the substantiallynonmagnetic physical barrier 1620 of FIG. 16 is replaced with a magneticphysical barrier 1710. FIG. 17 also shows that magnet 1616 of FIG. 16 isremoved from proximity to bottom substrate 1612. The magnetic physicalbarrier 1710 is used to (1) immobilize the magnetically responsive beads1618 and (2) to reduce the gap at the point of splitting, therebyassisting the droplet splitting operation. Additionally, because of theexistence of the rigid magnetic physical barrier 1710, consistentsplitting may be obtained substantially at the same point.

The process may include, but is not limited to, the following steps. InStep 1, the magnetic physical barrier 1710 immobilizes the magneticallyresponsive beads 1618 in the aqueous slug 1622. The aqueous slug 1622 isintersected by the magnetic physical barrier 1710, which reduces thegap. In Step 2, a droplet splitting operation is executed (e.g., formingdroplets 1624 and 1626), whereby the magnetically responsive beadsremain substantially immobilized by the magnetic physical barrier 1710and the magnetic physical barrier 1710 is used to reduce the gap at thepoint of splitting, thereby assisting the droplet splitting operation.In this example, magnetically responsive beads 1618 remain substantiallyimmobilized in droplet 1624 by magnetic physical barrier 1710 anddroplet 1626 is substantially free of beads 1618. The process may, forexample, be employed in a merge-and-split bead washing protocol.

7.7 Electrode Configurations for Improved Droplet Splitting

FIG. 18 illustrates embodiments of electrode configuration for improveddroplet splitting. In one example, FIG. 18A shows an electrode path 1810that includes a splitting region 1812 that includes a segmentedelectrode 1814, such as multiple electrode strips. In a splittingoperation, electrodes may be activated to extend a slug across theregion of electrode strips. The electorode strips may be deactivatedstarting with the outer strips and continuing to the inner strips inorder to cause a controlled split of the droplet at the electrode stripregion of the electrode path 1810. In an alternative embodiment, theelectrode strips may be rotated 90 degrees. In this embodiment,deactivation may start from the inner electrodes of the electrode stripsand continue to the outer electrodes in order to controllably split thedroplet at the electrode strips.

In another example, FIG. 18B shows an electrode path 1820 that includesa splitting region 1822 that includes a tapered electrode 1824 that mayspan a distance equivalent, for example, to about two standard dropletoperations electrodes. In operation, a droplet may be extended alongelectrodes of the electrode path across tapered electrode 1824.Electrode 1824 or the adjacent electrode 1825 may be deactivated tocontrollably split the droplet.

In yet another example, FIG. 18C shows an electrode pattern 1830 thatincludes a splitting region 1832 that includes a long tapered electrode1834 and a short tapered electrode 1836, where the smallest end of thetapered electrodes face one another. The tapered electrode pair may spana distance equivalent, for example, to about three standard dropletoperations electrodes. In operation, a droplet may be extended alongelectrodes of the electrode path across tapered electrodes 1834 and1836. Electrode 1834 and/or electrode 1836 may be deactivated tocontrollably split the droplet.

In yet another example, FIG. 18D shows an electrode pattern 1840 thatincludes a splitting region 1842 that includes a long tapered electrode1842 and a short interlocking electrode 1844, where the smallest end ofthe tapered electrode 1842 faces the interlocking electrode 1844. Theelectrode pair may span a distance equivalent, for example, to aboutthree standard droplet operations electrodes. In operation, a dropletmay be extended along electrodes of the electrode path across taperedelectrodes 1844 and 1846. Electrode 1844 and/or electrode 1846 may bedeactivated to controllably split the droplet.

In yet another example, FIG. 18E shows an electrode pattern 1850 thatincludes a splitting region 1852 that includes a segmented electrode1854, such as multiple row or columns of electrode strips. n operation,a droplet may be extended along electrodes of the electrode path acrosssplitting region 1852. Each segment may be independently deactivated asdesired to controllably split the droplet.

7.8 Improved Detection

A process for the detection of supernatant after adding a substrate tothe assayed magnetically responsive beads is disclosed, in accordancewith the invention. After the washing protocol to remove the excessunbound antibody is complete, a chemiluminescent substrate is added tothe assayed and washed beads, which produces chemiluminescence as aresult of the reaction between the enzyme on the beads and thesubstrate.

The substrate may be incubated with the magnetically responsive beadsfor some fixed time, where the magnetically responsive beads aresubstantially immobilized and the supernatant is transported away fordetection. This approach reduces, preferably entirely eliminates, theneed to transport the magnetically responsive bead droplet over longdistances to the detector and also reduces, preferably entirelyeliminates, the possibility of loss of beads during the transportoperation.

Alternatively the antibody-antigen-enzyme complex can be released fromthe bead by chemical or other means into the supernatant. The beads maythen be substantially immobilized and the supernatant processed furtherfor detection.

Additionally, the same split, merge, and transport strategies that areexplained for incubating beads/antibodies/sample mixture may be employedhere also for incubating substrate and assayed beads.

Bead based sandwich or competitive affinity assays, such as ELISAs, maybe performed using the procedures described in this application inconjunction with various steps described in International PatentApplication No. PCT/US06/47486, entitled “Droplet-Based Biochemistry,”filed on Dec. 11, 2006. Further, after incubation, unbound samplematerial and excess reporter antibody or reporter ligand may be washedaway from the bead-antibody-antigen complex using various dropletoperations. A droplet of substrate (e.g., alkaline phosphatasesubstrate, APS-5) may be delivered to the bead-antibody-antigen complex.During incubation, the substrate is converted to product which begins tochemiluminesce. The decay of the product (which generates light) issufficiently slow that the substrate-product droplet can be separatedfrom the alkaline phosphatase-antibody complex and still retain ameasurable signal. After an incubation period of the substrate with thebead-antibody-antigen complex (seconds to minutes), the magneticallyresponsive bead-antibody-antigen complex may be retained with a magneticfield (e.g., see U.S. patent application Ser. No. 60/900,653, filed onFeb. 9, 2007, entitled “Immobilization of magnetically-responsive beadsduring droplet operations,”) or by a physical barrier (e.g., see U.S.patent application Ser. No. 60/881,674, filed on Jan. 22, 2007, entitled“Surface-assisted fluid loading and droplet dispensing,” the entiredisclosure of which is incorporated herein by reference) and only thesubstrate-product droplet may be presented (using droplet operations) tothe sensor (e.g., PMT) for quantitation of the product.

The substrate-product droplet alone is sufficient to generate a signalproportional to the amount of antigen in the sample. Incubation of thesubstrate with the magnetically responsive bead-antibody-antigen complexproduces enough product that can be quantitated when separated from theenzyme (e.g., alkaline phosphatase). By measuring the product in thismanner, the bead-antibody-antigen complex does not have to be presentedto the PMT. There are no beads or proteins to “foul” the detector areaas they are never moved to this area. Also, the product droplet does nothave to oscillate over the detector to keep beads in suspension duringquantitation. The droplet volume may also be reduced in the absence ofbeads. Detection of the bead-antibody-antigen complex may employ a slugof liquid (e.g., 4 droplets) to move the complex, whereas with thebeadless method the droplet could be smaller (e.g., less than 4droplets). Time to result may also be shorter with this approach whenperforming multiplex ELISAs because the product droplet can be moved tothe detector more quickly in the absence of beads.

Bead based sandwich or competitive affinity assays, such as ELISAs, maybe performed using droplet operations for one or more steps, such ascombining sample, capture beads and reporter antibody or reporterligand. After incubation, unbound sample material and excess reporterantibody or reporter ligand may be washed away from thebead-antibody-antigen complex using an on-chip washing protocol. Afterwashing, a droplet of substrate (e.g., alkaline phosphatase substrate,APS-5) may be delivered to the bead-antibody-antigen complex. During theincubation, the substrate is converted to product which begins tochemiluminesce. The decay of the product (which generates light) issufficiently slow that the substrate-product droplet can be separatedfrom the alkaline phosphatase-antibody complex and still retain ameasurable signal. After an incubation period of the substrate with thebead-antibody-antigen complex (seconds to minutes), the magneticallyresponsive bead-antibody-antigen complex may be retained with a magnetor by a physical barrier and only the substrate-product droplet may bepresented (using droplet operations) to the sensor (e.g., PMT) forquantitation of the product.

The substrate-product droplet alone is sufficient to generate a signalproportional to the amount of antigen in the sample. Incubation of thesubstrate with the magnetically responsive bead-antibody-antigen complexproduces enough product that can be quantitated when separated from theenzyme (e.g., alkaline phosphatase). By measuring the product in thismanner, the bead-antibody-antigen complex does not have to be presentedto the PMT. There are no beads or proteins to “foul” the detector areaas they are never moved to this area. Also, the product droplet does nothave to oscillate over the detector to keep beads in suspension duringquantitation. The droplet volume may also be reduced in the absence ofbeads. Detection of the bead-antibody-antigen complex may employ a slugof liquid (e.g., 4 droplets) to move the complex, whereas with thebeadless method the droplet could be smaller (e.g., less than 4droplets). Time to result may also be shorter with this approach whenperforming multiplex ELISAs because the product droplet can be moved tothe detector more quickly in the absence of beads.

FIG. 19 illustrates detection strategies for quantifying an analyte. Inparticular, the immunoassay may be developed without any secondaryantibody that is labeled with enzyme, fluorophore, or quantum dots.After binding of the analyte to the antibody that is bound to themagnetically responsive beads, the hydrodynamic diameter of the beadsincreases due to the immune complex that is bound to the surface of thebead. A superconductive quantum interference device (SQUID) gradiometersystem may be used in order to measure the standard magnetization (Ms)of magnetically labeled immune complexes, such as the A₅-Ag complexshown in FIG. 19.

7.9 Operation Fluids

For examples of fluids that may be subjected to droplet operations usingthe approach of the invention, see the patents listed in section 2,especially International Patent Application No. PCT/US2006/047486,entitled, “Droplet-Based Biochemistry,” filed on Dec. 11, 2006. In someembodiments, the fluid includes a biological sample, such as wholeblood, lymphatic fluid, serum, plasma, sweat, tear, saliva, sputum,cerebrospinal fluid, amniotic fluid, seminal fluid, vaginal excretion,serous fluid, synovial fluid, pericardial fluid, peritoneal fluid,pleural fluid, transudates, exudates, cystic fluid, bile, urine, gastricfluid, intestinal fluid, fecal samples, fluidized tissues, fluidizedorganisms, biological swabs, biological washes, liquids with cells,tissues, multicellular organisms, single cellular organisms, protozoa,bacteria, fungal cells, viral particles, organelles. In some embodiment,the fluid includes a reagent, such as water, deionized water, salinesolutions, acidic solutions, basic solutions, detergent solutions and/orbuffers. In some embodiments, the fluid includes a reagent, such as areagent for a biochemical protocol, such as a nucleic acid amplificationprotocol, an affinity-based assay protocol, a sequencing protocol,and/or a protocol for analyses of biological fluids.

The fluids may include one or more magnetically responsive and/ornon-magnetically responsive beads. Examples of droplet actuatortechniques for immobilizing magnetically responsive beads and/ornon-magnetically responsive beads are described in the foregoinginternational patent applications and in Sista, et al., U.S. patentapplication Ser. No. 60/900,653, entitled “Immobilization ofMagnetically-responsive Beads During Droplet Operations,” filed on Feb.9, 2007; Sista et al., U.S. patent application Ser. No. 60/969,736,entitled “Droplet Actuator Assay Improvements,” filed on Sep. 4, 2007;and Allen et al., U.S. patent application Ser. No. 60/957,717, entitled“Bead Washing Using Physical Barriers,” filed on Aug. 24, 2007, theentire disclosures of which is incorporated herein by reference.

CONCLUDING REMARKS

The foregoing detailed description of embodiments refers to theaccompanying drawings, which illustrate specific embodiments of theinvention. Other embodiments having different structures and operationsdo not depart from the scope of the present invention. Thisspecification is divided into sections for the convenience of the readeronly. Headings should not be construed as limiting of the scope of theinvention. The definitions are intended as a part of the description ofthe invention. It will be understood that various details of the presentinvention may be changed without departing from the scope of the presentinvention. Furthermore, the foregoing description is for the purpose ofillustration only, and not for the purpose of limitation, as the presentinvention is defined by the claims as set forth hereinafter.

1. A method of splitting a droplet comprising a magnetically responsivebead, the method comprising: (a) providing a droplet actuatorcomprising: (i) a plurality of droplet operations electrodes configuredto transport the droplet; and (ii) a magnetic field present at theplurality of droplet operations electrodes; (b) immobilizing themagnetically responsive bead using the magnetic field; and (c) using theplurality of droplet operations electrodes to split the droplet intofirst and second droplets, wherein the magnetically responsive beadremains substantially immobilized.
 2. The method of claim 1 furtherwherein the splitting involves using a hydrophilic patch.
 3. The methodof claim 1 further comprising using a magnet to generate the magneticfield.
 4. The method of claim 1 further comprising using a magnetembedded within a gasket of the droplet actuator to generate themagnetic field.
 5. The method of claim 1 further comprising positioninga magnet proximate a gasket of the droplet actuator to generate themagnetic field.
 6. The method of claim 1 further comprising using aphysical barrier to facilitate splitting of the droplet.
 7. The methodof claim 1 further comprising using a magnetized physical barrier tofacilitate splitting of the droplet.
 8. The method of claim 1 furthercomprising positioning a magnetic shielding material in the dropletactuator to selectively minimize the magnetic field.
 9. The method ofclaim 1 wherein the magnetic field is sufficiently strong to hold themagnetically responsive beads substantially immobile during a dropletoperation.
 10. The method of claim 1 wherein the magnetic field issufficiently weak to enable the magnetically responsive bead to be movedaway from the magnetic field during a droplet operation.
 11. The methodof claim 1 wherein the first droplet contains at least substantially allof the beads and the second droplet is at least substantially lacking inbeads.
 12. The method of claim 1 wherein the droplet operationselectrodes comprise an electrode path having a droplet splitting region,the droplet splitting region including a segmented electrode comprisinga plurality of electrode strips, including inner electrode strips andouter electrode strips, wherein the electrode strips can beindependently activated and deactivated to cause the controlledsplitting of the droplet in the droplet splitting region.
 13. The methodof claim 12 wherein the droplet is split into first and second dropletsby: (i) activating the droplet operations electrodes to extend a dropletacross the electrode strips of the segmented electrode; and (ii) causingthe controlled splitting of the droplet in the droplet splitting regionby either deactivating the inner electrode strips followed bydeactivating the outer electrode strips, or by deactivating the outerelectrode strips followed by deactivating the inner electrode strips.14. The method of claim 1 wherein the droplet operations electrodescomprise an electrode path having a droplet splitting region, thedroplet splitting region including a tapered electrode that has a lengthalong the electrode path that is about twice that of an adjacent dropletoperations electrode.
 15. The method of claim 1 wherein the dropletoperations electrodes comprise an electrode path having a dropletsplitting region, the droplet splitting region including two adjacenttapered electrodes that have a combined length along the electrode paththat is about three times that of an adjacent droplet operationselectrode.
 16. The method of claim 1 wherein the droplet operationselectrodes comprise an electrode path having a droplet splitting region,the droplet splitting region including a segmented electrode comprisingmultiple rows and columns of electrode strips that can be independentlyactivated and deactivated to cause the controlled splitting of thedroplet in the droplet splitting region.
 17. The method of claim 1further comprising: (i) a bottom substrate having a droplet operationssurface comprising the droplet operations electrodes; (ii) a topsubstrate separated from the droplet operations surface to form a gap;and (iii) a physical barrier extending from the top substrate into thegap and constricting the gap in proximity with the one or more dropletoperations electrodes.
 18. The method of claim 17 wherein the barrierproduces a magnetic field.
 19. The method of claim 17 further comprisinga first electromagnet arranged near the top substrate of the dropletactuator and a second electromagnet arranged near the bottom substrateof the electromagnet.
 20. The method of claim 19 further comprisingusing the first electromagnet and second electromagnet to disperse themagnetically responsive beads within the droplet by switching on and offthe magnetic field produced by the first electromagnet and secondelectromagnet.